The overall objective of the proposed studies is to provide an understanding of the mechanism of insulin action. Specifically, the pathway by which insulin stimulates site specific protein phosphorylation in its target cell will be studied. When insulin is added to rat liver cells, an increase in levels of phosphorylation of a 46,000 mw phosphoprotein is observed. This protein is also phosphorylated by the cAMP-dependent protein kinase. However, evidence exists that phosphorylation occurs in a site specific manner. I propose to isolate the 46,000 mw phosphoprotein and map both the insulin-sensitive and cAMP-dependent protein kinase phosphorylation by enzymatic and chemical degradation. Antibody will be prepared to the purified phosphoprotein, and this antibody employed in the development of a highly specific assay for the protein kinase responsible for the insulin stimulated phosphorylation. The insulin-sensitive cAMP-dependent protein kinase will be isolated and its control mechanism determined. Knowledge of the mechanism of stimulation of this protein kinase may enable me to establish the events which occur immediately following insulin receptor interaction.